Determination of rope infection



Nov5,1935. H. H. BUNZELL Zw@ DETERMINATION OF ROPE INFECTION Filed May3l, 1930 L vc of INVENTOR.

Herben #5U/72e# A TTORNEYS.

Patented Nov. 5, 1935 UNITED STATES PATENT OFFICE 3 Claims.

This invention relates to determinations of conditions leading to thedevelopment of ropiness in bread and to apparatus adapted for suchdeterminations. Rope is an infection of bread by an 5 organism whosespores survive baking temperature and it is manifested by the odor o-fan infected loaf, the yellowish discoloration in the advanced stages,the silvery threads forming in the loaf when broken and the soft stickyand clammy crumb. This infection has long been the dread of the bakingfraternity for great losses may be incurred if the condition is notpromptly controlled.

As the general belief prevails that flour is the principal, if not theonly carrier of this infection,

a test has been worked out by Watkins for determining rope in flour(Journal Society of Chemical Industry 1906, p. 350). This test is inextensive use; but it requires a bacteriologist of considerable skill toavoid unintended infection of the material tested, and furthermore theappearance of the inoculated and incubated fingers of bread is not suchas to definitely show the presence or absence of rope in all but verypronounced cases.

For determining the presence of rope organisms in the baked product, themethods heretofore followed of detecting the physical or chemicalchanges through odor, feel and appearance have at least two seriousobjections. In the rst place usually two or three days is requiredbefore a definite conclusion can be reached. Secondly such methods arenot at all exact and therefore make it difficult toy differentiate in aquantitative sense between the infection-carrying power of differentours or other ingredients. For instance, if bread coming from a bakeryis to be tested for the purpose of evaluating conditions existing in abakery, these methods are not suficiently exact to indicate whether thecondition of infectedness is increasing or decreasing.

According to the present invention determinations of susceptibility torope can be made in the bread soon after baking as the method involvedis sufliciently sensitive to detect the presence of active organisms inthe incipient stages when neither odor nor feel will prove theirpresence with certainty. It lends itself to determining the possible'source of infection or whether susceptibility to infection exists in abakery, for I have found that other bread ingredients such as yeast ormilk or water, or the scrapings from diiferent part of machinery can bea means of rope infection'. As compared with the heretofore acceptedmethods of determining rope infection (Cl. 22E-230) it presents inaddition to the advantages of increased sensitiveness and accuracy, theadaptability to an expression of values in definite numbers to therebyenable the investigator to follow theenhancement or diminution of thecondi- 5' tion of rope infection'with considerable accuracy. Inpracticing the present invention, sample loaves are baked containing theingredient in question, care being taken to avoid contamination. If theflour is to be tested it is desirable 10 to reduce the ingredients to aminimum and biscuits using only the flour, water and baking powder arepreferably prepared; the baking powder is one that has previously beentested with flour known to be free from rope to insure its 15 safe use.The freshly baked bread samples or biscuits are then subjected toincubation at high humidity and at a temperature depending upon thelength of incubation; thus for periods of incubation of about 24, 48 and72 hours, a tempera- 20 ture of 30 C. is generally ample, while forshort periods about 50 C. will be found preferable.

Infected fresh bread or biscuits just from the oven exhibit no catalaseactivity but upon being subjected to incubation the catalase activitybe- 25 comes pronounced, and the extent of this activity isproportionate to the extent of ropiness. The measurement of catalaseactivity therefore becomes a measure of rope infection and it is thisprinciple that is the basis of this invention. 30

An apparatus for obtaining such catalase determinations is shown in theaccompanying drawing in which- Figure 1 is a front elevation of theapparatus shown suspended in a jar that is indicated in 35 y section;and

Figure 2 is a side elevation, but with the stop cocks turned indifferent positions from that illustrated in Figure l.

The apparatus is preferably made of glass and 40 includes a compartmentI0 with a side opening l I closed by a rubber stopper I2. An arm I3extends upwardly from the compartment I0 having an open end Ill closedby a stopper I5. A separatory funnel i6 with a turn` cock I'I has itstube 45 I inserted through the stopper I5. A tube I9 leads from the sideof the arm I3 and this` tube has an upturned end 2li. Another tube 2Ialso opens from the side of the arm I3, spaced approximately "V from thetube I9; the tube 2l 50 is prolonged and bent to form a mano-meter 22with the legs 23 and 24 graduated in millimeters.

The funnel It has its open upper end 25 closed by a rubber stopper 26. Atube 21 extends from the Side of the funnel I6 and attached thereto 55is a three-way stop cock 28. From the stop cock there extends a venttube 29 opening to the atmosphere and a third tube 3G having adownwardly extending end 3|. When the compartment and the funnel areassembled, as shown in the drawing, the end 3l is in alignment with theupturned end 20 of the tube I9 so that the two ends can be connected bya piece of rubber tubing 32; (the tubing is omitted from Fig. 1 for sakeof clearness) For securing the parts in assembled relation, the arm I3is provided with glass hooks 33; rubber bands 34 are snapped about thesehooks over the turn cock I1.

The bend 35 connecting the tube 2| with the leg 23 of the manometer ispreferably so located that a glass rod 36 can be passed through it andunder the tube 21 whereby the assembled apparatus can be suspended in aWater bath contained in a jar 31.

To use the apparatus for rope determination in bread the two separableparts are assembled as shown. As an illustration about 25 grams of breadpulp are ground with 50 cc. of tap Water and made up to cc. withadditional Water. By holding the apparatus at a slant with the openingII of compartment I0 tilted upwardly, the fluid mass can be poured intothe compartment; remaining particles of pulp are flushed Y into theapparatus with 10 cc. of water, after which the stopper I2 is insertedtightly. The turn cock I1 is closed as shown in Fig. 2 while thethree-way cock 28 is set to permit escape or entrance of air as shown inFig. 1. The apparatus is then suspended in the jar 31 filled to about aninch from the top with water that should be approximately at roomtemperature (within 1 F.) so that its temperature will not changeappreciably during a period of 15 to 20 minutes. The glass rod 36supporting the apparatus can be supported on the sides of the jar 31.The apparatus is allowed to remain in the bath for about 10 minutes toestablish uniform temperature conditions and during this interval about10 cc. of 3% hydrogen'peroxide is introduced into the funnel I6. Mercuryis contained in the graduated manometer arms to the zero marks. Therubber stopper 26 is inserted into the funnel.

After the temperature has become equalized the stop cock 28 is turned tocut off communication with the outside but so as to establishcommunication through the tubes 21 and 30 and rubber tubing 32 to thearms I3. The stop cock I1 is now opened to allow the hydrogen peroxide ssolution to flow into compartment I0. If the cornpartment has a capacityof 100 cc. the introduction of 95 cc. of liquid leaves an air bubble of5 cc. to facilitate agitation. The apparatus is given a gentle shake ortwo and the time noted.

Readings are made in millimeters on the manometer at intervals of oneminute for ve minutes.

The operation of the apparatus depends upon the amount of oxygenliberated and this is readily calculated from the gas space, the changeof pressure as indicated by the manometer and the temperature; thebarometric pressure may be safely disregarded, as this introduces but aminor and insignificant error. Any unit may be selecte-d as for examplethe liberation of 1 microgram of perature t occupiesor v 273-l-t .0007273 cc.

In 40 cc. this amount of oxygen creates at 0 C. an additional pressureof .0133 mm. If 25 grams of bread are used this pressure represents,according to the definition suggested, one twentyfifth of a unit ofinfection; or one unit therefore theoretically produces at 0 C. in 5minutes a pressure of .3325 mm. At 20 C. each unit of infection producesa pressure of .367 mm. If 25 grams of bread produces too great areading, i. e. over mm., the amount of bread tested should be reduced toone-tenth or possibly one-hundredth and the results multipliedaccordingly.

In the incubation of the bread samples humidity is required solely toinsure propagation of the organisms present. For this reason highhumidity as previously stated is preferred; but the amount of moisturenormally present in the samples is generally suiiicient for thispurpose, particularly if care is taken to prevent evaporation as bywrapping each Ysample in wax paper. Following incubation each sample istested and the number of units of infection Idetermined in accorda-ncewith the above described procedure. The unit is of course based on anarbitrary selection of conditions; for example in the precedingparagraph the unit selected is the liberation of 1 microgram of oxygenper gram of bread that has been incubated at 50 C. for 24 hours; otherconditions can obviously be substituted. The units of infection sodetermined indicate the amount of rope preventative (generally vinegaror other acid) to be included in the bread dough for complete inhibitionof the infection. As it is well recognized that such preventatives donot otherwise add to the value of the bread but to the contrary aredisadvantageous, the procedure given affords a means of determiningwhether the preventative need be added at all; and if so the minimumamount required for complete prevention of rope.

I claim:

1. Method of determining rope infection in bread which comprisesincubating freshly baked bread and measuring the catalase activity ofthe incubated bread.

2. Method of determining rope infection in bread which comprisesincubating freshly baked bread, forming a pulp of the incubated bread inwater, adding hydrogen peroxide to the pulp, and measuring the amount ofoxygen liberated during a period of time.

3. Method of determining the degree of rope infection in bread whichcomprises subjecting the bread to incubation at high humidity, forming apulp of the incubated bread in water, adding hydrogen peroxide to thepulp, and measuring' the liberated oxygen.

HERBERT H. BUNZELL.

